The Potential Fungal Pathogens of Euonymus japonicus in Beijing, China

Euonymus japonicus tolerates the dry and frigid climate of Beijing, China, and effectively filters out particles during the winter. However, fungal infestation frequently causes extreme illness and can even lead to shrub death. In this study, 104 diseased E. japonicus specimens were collected from seven districts in Beijing. Seventy-nine isolates were identified as 22 fungal species in seven genera. The species were Aplosporella hesperidica, A. javeedii, A. prunicola, Botryosphaeria dothidea, Colletotrichum aenigma, Co. euonymi, Co. euonymicola, Co. gloeosporioides, Cytospora ailanthicola, C. albodisca, C. diopuiensis, C. discotoma, C. elaeagni, C. euonymicola, C. euonymina, C. haidianensis, C. leucostoma, C. sophorae, C. zhaitangensis, Diaporthe eres, Dothiorella acericola, and Pestalotiopsis chaoyangensis. On the basis of morphological and phylogenetic analyses, Colletotrichum euonymi, Co. euonymicola, Cytospora zhaitangensis, and Pestalotiopsis chaoyangensis were introduced as novel species. Colletotrichum euonymi, Co. euonymicola, and Pestalotiopsis chaoyangensis were subsequently confirmed as pathogens of E. japonicus leaves by pathogenicity testing. This study provides an important assessment of the fungi associated with diseases of E. japonicus in Beijing, China.


Introduction
The family Celastraceae, which includes 96 genera and over 1350 species, is extensively distributed in tropical, subtropical, and temperate climates as evergreen or deciduous trees, shrubs, or vine shrubs [1]. Euonymus japonicus, also called Japanese spindle, is one of the most prevalent and important species in Celastraceae in northern Chinese cities such as Beijing, the capital of China [2]. As a species of evergreen shrub, E. japonicus has strong resistance to the dry and cold conditions in Beijing and can efficiently reduce particulate matter in winter [2]. Furthermore, roots, stems, and leaves of the shrub have a high capacity to enrich heavy metals [3]. However, Japanese spindle in Beijing is frequently extremely ill and even dies because of fungal infestation ( Figure 1).
Fungi from many different taxa are associated with diseases of the same host plant species or genus. Raza et al. [4] described one new genus and 32 new species of culturable fungi associated with sugarcane disease in southern China, and Crous et al. [5] described seven new genera and 15 new species as foliar fungal pathogens of eucalypts. Accurate identification of pathogenic fungi provides a good theoretical foundation for the control of plant diseases. However, because fungi associated with fungal diseases of Japanese spindle in Beijing have not been systematically and extensively studied, effective disease prevention is difficult. Therefore, the variety of fungal species associated with Japanese spindle diseases in Beijing was examined in this study. In the study, 79 fungal isolates were classified as seven genera (Aplosporella, Botryosphaeria, Collectotrichum, Cytospora, Diaporthe, Dothiorella, conidia with colored cells in the middle three cells and colorless apical cells, and one to several apical appendages). Pestalotiopsis is a well-known phytopathogenic genera. Jiang et al. [39] introduced 10 new species of Pestalotiopsis from Fagaceae leaves in China. Pestalotiopsis breviseta, P. caroliniana, P. clavata, P. diospyri, P. gracilis, P. neglecta, and P. planimi have been recorded on host Euonymus [40][41][42][43].
During investigations of the diversity of fungal species that cause diseases of E. japonicus, several ascomycetous taxa associated with various disease symptoms were collected in Beijing. The objectives of this study are to (1) investigate fungal diseases on E. japonicus in Beijing, (2) identify the fungal species isolated from E. japonicus, and (3) test the pathogenicity of the novel species identified.

Sampling and Isolation
During 2020 to 2021, a survey was conducted in seven districts (Chaoyang, Daxing, Fengtai, Haidian, Mentougou, Shijingshan, and Xicheng) in Beijing, China. A total of 104 specimens (67 branches and 37 leaves) affected with different symptoms were collected. Isolates from leaves were obtained using tissue isolation methods. Leaf spots were cut into small pieces (0.2 × 0.2 cm) and placed on potato dextrose agar (PDA, 200 g potato, 20 g glucose, 20 g agar, and 1000 mL water) plates and incubated at 25 • C after surface sterilisation (1 min in 75% ethanol, 3 min in 1.25% sodium hypochlorite, then rinsed in distilled water and blotted on dry sterile filter paper). Fruiting bodies on diseased branches were shaved off the surface with a sterile blade after surface sterilisation, then the mucoid spore mass from conidiomata was put onto a PDA culture medium and incubated at 25 • C in darkness until spores germinated. Single germinating conidia were transferred onto fresh PDA plates. Hyphal tips were cut and transferred to a new PDA plate twice to obtain a pure culture for further study. Specimens are preserved at the working collection of X.L. Fan (CF) housed at the Beijing Forestry University (BJFC). Cultures of taxonomic novelties are deposited at the China Forestry Culture Collection Centre (CFCC).

Phylogenetic Analyses
The sequence datasets used in this study were based on Lin et al. [51,52] for Cytospora, Liu et al. [16] for Colletotrichum, Jiang et al. [39] for Pestalotiopsis, and Zhang et al. [6] for Botryosphariales, deleting the overly repetitive isolates of the same species and supplementing them with other sequences obtained from GenBank (Table S1). Sequence alignments of the individual loci were performed in MAFFT v. 6 [53] and adjusted by MEGA v. 6.0 [54]. Ambiguous regions were excluded from alignments. For the genus Colletotrichum, the ITS tree, including 15 species complexes, was first used for inferring delimitation to the species complex level before multi-locus phylogenetic analyses. Maximum Likelihood (ML) and Bayesian Inference (BI) were used for phylogenetic analyses of both each individual loci and the concatenated genes alignments. ML and BI analyses were computed using PhyML v. 3.0 [55] and MrBayes v. 3.1.2 [56], respectively. For ML analyses, GTR + GAMMA model of site substitution with 1000 bootstrap was set. For BI analyses, the best-fit evolutionary models for each partitioned locus were estimated in MrModeltest v. 2.4 [57]. BI analyses with a four simultaneous Markov Chain Monte Carlo (MCMC) were computed from random trees for 1,000,000 generations and sampled every 1000 generations, and the burn-in was set to 0.25. The resulting trees were viewed in Figtree v. 1.3.1 [58]. The multi-locus sequence alignments were deposited in TreeBASE 29991.

Morphology
For the isolates isolated from diseased branches, the fruiting bodies on the specimens corresponding to the isolates were used for morphological observation. For the isolates isolated from leaf spots, reproductive structures formed on PDA were used for morphological observation. The structure and size of conidiomata were photographed using the Leica stereomicroscope (M205 FA) (Leica Microsystems, Wetzlar, Germany). Over 30 conidiomata were sectioned, and 50 conidia were selected randomly to measure their lengths and widths using a Nikon Eclipse 80 i microscope (Nikon Corporation, Tokyo, Japan) equipped with a Nikon digital sight DS-Ri2 high-definition color camera with differential interference contrast (DIC). Colony color on PDA were described according to the color charts of Rayner [59].
Branches were cut to 20-cm lengths, with the bottoms submerged in water and tips sealed with sealing film. To inoculate branches, a hole punch with 5-mm diameter and approximately 0.5-mm thickness was used to scald branches 10 cm from the tip. A 14-day culture block of the same size was attached to the wounds. Branches were then wet with skimming cotton moistened with sterile water and covered in sealing film. To inoculate leaves, a sterile inoculation needle pierced the leaves five to seven times, and 4-mmdiameter 14-day culture blocks were placed on the wounds. Branch and leaf inoculations were incubated at 25 • C and 70% humidity. Six replicates were prepared for each isolate, and a sterile PDA plug served as the control. Experiments were conducted twice. To fulfil Koch's postulates, re-isolations were made from lesions to compare the morphological features and DNA sequences with those of the original isolates. R 4.2.2 with the packages "ggplot2" [60] and "ggpubr" [61] was used to analyse pathogenicity data and output figure. Data were analysed using Tukey's honestly significant difference (HSD) test (α = 0.05) by one-way analysis of variance (ANOVA).

Phylogenetic Analyses
The best-fit models used in Bayesian analyses and the statistics of ML trees are presented in Tables 2 and 3, respectively. The ML trees with ML bootstrap support values and posterior probabilities are shown in Figures 3-5 and S1-S5. The results of Bayesian analyses did not significantly differ from those of ML trees.

Phylogenetic Analyses
The best-fit models used in Bayesian analyses and the statistics of ML trees are presented in Tables 2 and 3, respectively. The ML trees with ML bootstrap support values and posterior probabilities are shown in Figures 3-5 and S1-S5. The results of Bayesian analyses did not significantly differ from those of ML trees.    from BI. Ex-type isolates are in bold. Isolates highlighted with blue colours were obtained in this study.  . Phylogram of Cytospora based on maximum likelihood (ML) analysis of the dataset of combined ITS, act, rpb2, tef1-α, and tub2 genes. ML bootstrap support values above 70% are shown near nodes. Thickened branches represent posterior probabilities above 0.95 from BI. Ex-type isolates are in bold. Isolates highlighted with blue colours were obtained in this study.

Figure 5.
Phylogram of Pestalotiopsis based on maximum likelihood (ML) analysis of the dataset of combined ITS, tef1-α, and tub2 genes. ML bootstrap support values above 70% are shown near Figure 5. Phylogram of Pestalotiopsis based on maximum likelihood (ML) analysis of the dataset of combined ITS, tef1-α, and tub2 genes. ML bootstrap support values above 70% are shown near nodes. Thickened branches represent posterior probabilities above 0.95 from BI. Ex-type isolates are in bold. Isolates highlighted with blue colours were obtained in this study.

Taxonomy
Aplosporella prunicola Damm  Notes: In this study, four isolates CFCC 55550-55552 and 57541 were grouped together with Aplosporella prunicola and A. yalgorensis in phylogenetic analyses with 98 ML bootstrap support value and 0.98 posterior probabilities. The four isolates in this study can be distinguished from A. yalgorensis based on ITS and tef loci (for 10-11/520 bp in ITS, 2-6/317 bp in tef ). In ML tree, CFCC 55550-55551 clustered with A. prunicola with 83 ML bootstrap support value with 100% repetitive ITS sequences. CFCC 55552 and 57541 are only one base different from A. prunicola in ITS region. Additionally, the conidia size of CFCC 55552 on PDA are 16.5-21.5 × 10.0-10.5 µm, which is consistent with the morphological characteristics of ex-type of A. prunicola for (17)  Notes: Cesati and De Notaris [71] first introduced the genus Botryosphaeria with 12 species described. However, they did not specify a type species of this genus. Barr et al. [72] suggested that Botryosphaeria dothidea (Basionym: Sphaeria dothidea Moug.: Fries [73]) should be a lectotype of this genus. Slippers et al. [74] re-examined that the host of the holotype of Sphaeria dothidea in the Fries herbarium was Rosa sp., which was not consistent with the description of Fries [73] (on Fraxinus sp.). Additionally, the only other specimen identified as S. dothidea on Fraxinus sp. in the Fries herbarium was immature with no spores [74,75]. This specimen was designated as a neotype [74]. Then, Slippers et al. [74] re-collected specimens from a nearby locality and designated an epitype (PREM 57372) on Prunus sp. collected from Crocifisso, Switzerland, with an ex-epitype culture (CBS 115476 = CMW 8000) with phylogenetic data. Zhang et al. [6] reduced four species to synonymy with Botryosphaeria dothidea based on the high sequence similarity values in ITS region. In this study, twenty-one isolates clustered together with B. dothidea in ML and BI trees. Therefore, they are identified as Botryosphaeria dothidea.
Cytospora discostoma M. Pan  Notes: Cytospora discostoma was first discovered on branches of Platycladus orientalis at Mentougou District in Beijing [83]. In this study, one isolate, CFCC 56276, clustered in a well-support clade (ML/BI = 100/1) with C. discostoma CFCC 53137 and 54368. The specimen BJFC CF20220122 in this study was collected from branches of Euonymus japonicus at Mentougou District in Beijing, where Cytospora discostoma was first discovered.
Descriptions: See Shang et al. [84]. Notes: Cytospora diopuiensis was discovered on bark of dead wood in Thailand [84]. Jiang et al. [85] reported this species on Kerria japonica f. pleniflora in China. In this study, two isolates from leaves of Euonymus japonicus and two isolates from branches clustered in a well-supported clade with C. diopuiensis (ML/BI = 100/1). Therefore, they were identified as Cytospora diopuiensis. Notes: Cytospora euonymicola was first introduced on Euonymus kiautschovicus in Shaanxi Province, China [32]. In this study, two isolates grouped together with C. euonymicola in ML and BI trees (ML/BI = 100/1). Morphologically, the conidia size in this study were similar in C. euonymicola described by Fan et al. [32] (4.5-5.0 × 1.0-1.5 µm vs. 4-5 × 1 µm). Therefore, the two isolates in the current study are identified as Cytospora euonymicola based on phylogeny and morphology.

Pathogenicity Test
In the leaf inoculation assays, fourteen days after inoculation, leaf lesions were caused by all three species isolated from leaves (Co. euonymi CFCC 55542, Co. euonymicola CFCC 55486, and P. chaoyangensis CFCC 55549) (Figures 10 and 11, Table 4). Disease sites initially turned yellow, and as disease progressed, diseased patches enlarged and took on a waterstained appearance, ultimately leading to wilting and consequent death. No symptoms were observed in the non-inoculated controls. All pathogenic species were re-isolated from lesions or conidia masses of inoculated leaves.

Pathogenicity Test
In the leaf inoculation assays, fourteen days after inoculation, leaf lesions were caused by all three species isolated from leaves (Co. euonymi CFCC 55542, Co. euonymicola CFCC 55486, and P. chaoyangensis CFCC 55549) (Figures 10 and 11, Table 4). Disease sites initially turned yellow, and as disease progressed, diseased patches enlarged and took on a water-stained appearance, ultimately leading to wilting and consequent death. No symptoms were observed in the non-inoculated controls. All pathogenic species were reisolated from lesions or conidia masses of inoculated leaves.
In the branch inoculation assay, no symptoms were observed with C. zhaitangensis inoculation or in the non-inoculated controls.

Discussion
Euonymus japonicus is an evergreen shrub that often becomes seriously diseased and even dies from fungal infestations in Beijing, China. In the current study, 79 isolates were obtained from 104 specimens collected from seven districts in Beijing City. The isolates included 22 species in seven genera, which were Aplosporella (eight isolates, three species), Botryosphaeria (21 isolates, one species), Colletotrichum (10 isolates, four species), Cytospora (31 isolates, 11 species), Diaporthe (three isolates, one species), Dothiorella (four isolates, one species), and Pestalotiopsis (two isolates, one species). Among the 22 species, Co. euonymi, Co. euonymicola, C. zhaitangensis, and P. chaoyangensis were identified as novel species on the basis of morphological and phylogenetic analyses. Colletotrichum euonymi, Co. euonymicola, and P. chaoyangensis were confirmed as pathogens on leaves of E. japonicus. In this study, A. hesperidica, A. javeedii, A. prunicola, Co. aenigma, C. ailanthicola, C. albodisca, Figure 11. Average lesion diameter (mm) resulting from inoculation with Euonymus japonicus. Vertical bars represent standard error of means. Different letters above the bars indicate treatments that were significantly different (α = 0.05).
In the branch inoculation assay, no symptoms were observed with C. zhaitangensis inoculation or in the non-inoculated controls.
Cytospora had the highest diversity of species associated with E. japonicus (11 species). The genus includes numerous important pathogens and saprophytic fungi on various hosts [32,33,83,96]. Branch and stem diseases frequently result in skin rot, dryness, and plant death [51,52,57,97]. Among the 10 known species obtained from E. japonicus in the current study, C. ailanthicola and C. haidianensis are confirmed pathogens on Populus and Euonymus, respectively [32,52]. However, C. zhaitangensis, the novel species isolated from branches, was not pathogenic on E. japonicus. The pathogenicity of other Cytospora species on Euonymus needs to be studied further.
Botryosphaeria dothidea was the species with the highest number of isolates (21 isolates), which were distributed in Chaoyang, Daxing, Haidian, Mentougou, Shijingshan, and Xicheng districts. Chaoyang District had the most fungal species occurring on E. japonicus (11 species in seven genera), followed by Haidian and Mentougou districts (nine species in five and four genera, respectively). The differences among districts could be because Chaoyang District is a major industrial district in Beijing, which has more environmental pollution. Schmidt et al. [98] concluded that Sordariomycetous fungi dominated at the polluted site and species diversity of endophytes was higher at the unpolluted site. These changes could weaken plants and increase susceptibility to disease.
Multiple infections can occur on different plant parts. For example, B. dothidea causes apple ring rot of stems, twigs, and fruits [99], and Co. gloeosporioides causes anthracnose of leaves and fruits [100]. Among the seven genera in this study, Aplosporella, Botryosphaeria, Cytospora, Dothiorella, and Diaporthe are pathogenic and cause canker and dieback disease of various hosts [6,29,32,64,69,94,96]. However, eight isolates were obtained from leaves in the current study, i.e., B. dothidea CFCC 55575, C. diopuiensis CFCC 54692 and 55479, C. elaeagni CFCC 55477, C. euonymina CFCC 55524 and 55525, C. sophorae CFCC 55523, and Do. acericola CFCC 55559. The isolates also created reproductive structures on leaves, which indicated that the species may be able to infect branches as well as leaves. The pathogenicity of these species to leaves and stems needs to be studied further. Increased understanding of pathogen diversity is beneficial to E. japonicus production and maintenance because impacts of disease can be minimized, and disease management needs to be improved.

Supplementary Materials:
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/jof9020271/s1, Figure S1. Phylogram of Aplosporella based on maximum likelihood (ML) analysis of the dataset of combined ITS and tef1-α genes; Figure S2. Phylogram of Botryosphaeria based on maximum likelihood (ML) analysis of the dataset of combined ITS, tef1-α, and tub2 genes; Figure S3. Phylogram of Colletotrichum based on maximum likelihood (ML) analysis of the dataset of ITS gene; Figure S4. Phylogram of Diaporthe based on maximum likelihood (ML) analysis of the dataset of combined ITS, cal, his3, tef1-α, and tub2 genes; Figure S5. Phylogram of Dothiorella based on maximum likelihood (ML) analysis of the dataset of combined ITS, tef1-α, and tub2 genes; Table S1: Strains used in the molecular analyses in this study.

Data Availability Statement:
Alignments generated during the current study are available in Tree-BASE (accession http://purl.org/phylo/treebase/phylows/study/TB2:S29991 (accessed on 15 February 2023)). All sequence data are available in NCBI GenBank following the accession numbers in the manuscript.